Sterilization and disinfection processes, and the verification of the efficacy of such processes, are vital to the protection of the health and welfare of the general population. For example, sterilization and disinfection processes are of paramount importance in healthcare applications, food service applications, and even in some military and civil defense applications involving weapons of biological warfare. Sterilization processes are commonly applied to medical devices and equipment, instruments, food, water, containers for food and/or medical devices, laboratory spaces, hospital facilities, military and governmental facilities, and other physical spaces or articles where unwanted and potentially infectious pathogens may come in contact with human beings or other animals, causing infection. Because of the importance of such procedures, it is necessary to monitor the efficacy of each sterilization or disinfection procedure, to ensure that undesirable pathogens have been eliminated.
Sterilization is commonly understood to mean that, upon completion of the sterilization process, the treated article or space is characterized by a complete absence of viable microorganisms. “Disinfection,” in contrast, is indicative of processes used to reduce the level of pathogens in an area or on an article to a degree such that they would pose minimal risk of infection to a healthy person. Disinfection methods include application of hydrogen peroxide, ethanol, or chlorine bleach. Commonly used methods of sterilization include “hot” or heat-based methods, such as sterilization by dry heat or sterilization by moist heat (steam) and “cold” or low temperature methods, such as sterilization by ethylene oxide, peracetic acid, formaldehyde, gas plasma sterilization, e.g., using a hydrogen peroxide plasma, and radiation, such as gamma radiation or E-beam radiation.
Conventional practice, particularly in the area of medical and scientific devices, has been to accomplish the sterilization process by use of moist heat (most commonly using autoclave equipment). In more recent practice, particularly in the healthcare industry where implements and devices are becoming more and more delicate and made of diverse materials, such as plastics, low temperature sterilization processes are favored. For example, sterilization using gas plasmas and/or ethylene oxide is routinely used. Specialized sterilization equipment has been developed for use of these sterilization procedures. For example, the STERRAD® System (Advanced Sterilization Products, a Johnson & Johnson Company (Irvine, Calif.)) uses hydrogen peroxide vapor low temperature gas plasmas to sterilize medical devices.
It is necessary to monitor or evaluate the efficacy of processes used to sterilize or disinfect in order to assure that the sterilized equipment is safe for use. Commonly used means of monitoring the sterilization process(es) is by use of a sterilization process indicator. The sterilization process indicator is placed in close proximity to the products, articles, and/or in the space which is to be sterilized, and is subjected to the same sterilization procedure.
In general, there are two types of sterilization process indicators: (i) physical/chemical sterilization process indicators; and (ii) biological sterilization process indicators. A physical/chemical process indicator is used to measure directly or indirectly the adequacy of the physical sterilization conditions during the sterilization process (such as temperature, pressure, and/or contact with a specific chemical). For example, a physical/chemical sterilization process indicator may be formulated to change from a dark green to a bright green when the indicator has been subjected to a specific high temperature for a specified amount of time. By observing the change from dark green to light green, the person carrying out the sterilization procedure is assured that at least the temperature process parameter of the procedure is met, and may extrapolate that all pathogens present on the articles subjected to the process have been destroyed. However, physical/chemical sterilization process indicators verify the presence or absence of certain physical or chemical conditions, and therefore only indirectly reflect upon the viability or non-viability of pathogenic organisms present. They are not a direct measure of the survival or destruction of any bacteria or pathogens initially present on the articles or in the space.
In contrast, biological sterilization process indicators permit a more direct assessment of the viability or non-viability of a living organism subjected to the sterilization procedure. Biological sterilization process indicators or biological indicators (BIs) generally consist of a known number of microorganisms of known resistance to the selected mode of sterilization (“the indicator organism”), in or on a carrier, and enclosed in a protective package. The biological sterilization process indicator, like the physical/chemical process indicator, is subjected to the same sterilization processes of the article and/or space to be sterilized and, upon completion of the sterilization procedure, the viability/non-viability of the organisms is assessed through various means.
When using a BI, the degree of sterilization or disinfection may conventionally be expressed in terms of “log kill”—the number of orders of magnitude by which the known population of indicator organism is decreased by the sterilization/disinfection process. Under present FDA regulations (21 C.F.R. § 800 et seq.), a six log reduction (“6 log kill” or Sterility Assurance Level (SAL) of 10−6) is considered to be sufficient assurance that “sterilization” has been accomplished for medical devices intended to come in contact with breached skin or compromised tissue. A different log reduction may apply, depending on the intended use of the device or object to which the process has been applied.
Because the resistance or susceptibility of the indicator organism will necessarily influence the sterility assurance analysis, the indicator organism is selected to be more resistant to the chosen sterilization technique than the microbial, fungal, or viral population anticipated to be present on the non-sterile devices or in the non-sterile space. The resistance to sterilization is conventionally indicated by the D value or the Z value of a given organism under specific sterilization conditions. The D values and Z values of a given organism are determined in accordance with the published guidelines of the United States Pharmacopecia (USP).
Because of their known D values, commonly used indicator organisms include Bacillus stearothermophilus (for steam/moist heat sterilization procedures), Bacillus subtilis var. niger (ethylene oxide, hydrogen peroxide, or dry heat), Bacillus pumilus (radiation). Also commonly used are bacteria of the genus Clostridium (Clostridium sporogenes), Candida albicans, Aspergillus niger, Micrococcus luteus, Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli, as well as those organisms classified in Group 18, in Bergy, et al., Bergy's Manual of Determinative Bacteriology, 9th ed., Lippincott, William & Wilkins, 1999, the contents of which are incorporated herein by reference. In conventional practice, the indicator organism may be vegetative cells or endospores (spores).
A standard type of biological sterilization process indicator is a device containing or including a known population of bacterial spores. The indicator is placed into a sterilization chamber (or at the site of sterilization or disinfection) and subjected to a sterilization process, along with the objects or articles to be sterilized or disinfected. Subsequent to the completion of the sterilization procedure, the indicator spores are contacted with a sterile growth medium and incubated for a selected period under conditions which favor germination of the spores and proliferation of vegetative cells. Growth of bacterial cells, determined by, for example, the presence or absence of certain metabolic products, or by observation of plated culture suspensions, indicate that the sterilization process was insufficient to destroy all of the spores, and therefore, that the process may not have achieved suitable sterilization or disinfection of the articles that accompany the indicator through the process. Although a wide variety of devices for containing the spores of the biological indicator has been developed, there are few variations in the general process of assessing the viability of the indicator organism following completion of the sterilization process. All involve an observation of the presence or absence of bacterial growth, post sterilization process.
Many biological indicators are self contained, in that they comprise spores and/or vegetative bacterial cells and germination/culture medium in a single container, typically in separate compartments. Following sterilization, the spores are combined with the medium, and the entire container is incubated in order to allow for the detectable growth to occur. Other known biological indicators comprise spores disposed in or on a carrier. After being exposed to the sterilization process, the carrier is contacted with a germination/culture medium to allow detectable growth from the spores to occur.
Like the physical/chemical sterilization process indicators, conventional BIs have several drawbacks. First, use of the conventional biological sterilization assurance process does not allow one to rapidly determine whether the sterilization process to which the indicator has been subjected was sufficient to destroy an adequate number of the spores in the indicator, and therefore does not permit rapid evaluation of the efficacy of the sterilization procedure. Because the spores of biological indicators require that the viability assessments be accomplished by permitting sufficient time such that the growth or lack of growth of the indicator organism can be assessed, rapid turn around time of, for example, medical devices, is impossible. In most cases, the incubation time required for a viability assessment is approximately forty-eight hours. During the time that the viability of the indicator organisms is being assessed for growth, the sterilized articles cannot be used safely.
In smaller facilities, such as outpatient clinics, which often lack microbiology labs, the organisms of the BIs must be sent to other facilities for cultivation and viability assessment after application of the process, further adding to the delay and costs in obtaining results. Many healthcare facilities have limited resources; they must reuse their sterilized instruments as soon as possible, preferably immediately or soon after sterilization or disinfection. Thus, the delay between sterilization and confirmation of sterility or sterility assurance is often expensive and impractical. Further, during and after the indicator organism is being cultured, accurate results rely on the maintenance of a sterile atmosphere and consistent practice of aseptic technique on the part of laboratory technicians. Hence, the assessment process is susceptible to human error. A need in the art exists for a more rapid method of assessing the efficacy of a sterilization procedure.
Prior art attempts have been made to overcome the time delay inherent in the use of biological sterilization process indicators. For example, a system has been developed that correlates sterilization efficacy with the activation (or deactiviation) of one or more thermostable enzymes present in the indicator organism. However, such systems provide again, only an indirect confirmation of sterility, and further, are not useful in connection with other non-heat based or “cold” sterilization methods, which would not serve to reliably deactivate thermostable enzymes. Additionally, because the outcome of sterility assurance tests based on evaluation of the inactivation of thermostable enzymes requires detection of a negative result (the absence of enzyme activity), it is fraught with potential errors. For example, inactivation of enzyme activity can have multiple causes, such as errors in assay performance (human error, technical failures), deficiency in enzyme substrate, or inactivation of the enzyme attributable to a cause other than the sterilization procedure.
Most commonly, the indicator organism selected for use in a biological process sterilization indicator is a bacterial endospore. Spores are preferred because they exhibit, overall through all species, a greater resistance to various sterilization methods, including heat sterilization, chemical sterilization (wet or plasmas), and radiation sterilization, and therefore always have greater D and/or Z values than their vegetative cell counterparts.
Dipicolinic acid (“DPA”; pyridine-2,6-dicarboxylic acid) is a component of bacterial spores, including spores of the genus Bacillus. Dipicolinic acid is represented by the structure:
In nature, DPA is present in spores as a substantially insoluble calcium salt (calcium dipicolinate), and is released upon the germination of the spore. While not wishing to be bound by theory, it is believed that DPA is present in the cortex and coat of the bacterial spore in an amount of about 10% to about 15% of total spore weight, and is present primarily in the form of calcium dipicolinate.
As it is not present in vegetative, non-sporulating bacterial cells, DPA has been suggested in the art as an indicator for the presence and quantification of bacterial spores (Hindle, et al., 1999, Analyst 124:1599–1604; U.S. Pat. No. 5,876,960). It has been recognized that the release of DPA from spores occurs after heat-induced loss of viability of the spores (Mallidis, et al., 1985, J. Appl. Bacteriol. 59:479–486) and upon germination of the spores (Scott, et al., 1978, J. Bacteriol. 135:133–137). However, other processes or conditions which induce the release of DPA from the spores have not been elucidated in the art, and no correlation between the release of DPA and the destruction of the spore has previously been disclosed.